Research ArticleVIROLOGY

A tripartite ssDNA mycovirus from a plant pathogenic fungus is infectious as cloned DNA and purified virions

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Science Advances  03 Apr 2020:
Vol. 6, no. 14, eaay9634
DOI: 10.1126/sciadv.aay9634
  • Fig. 1 Characterization of FgGMTV1 infecting F. graminearum strain HB58 and composition of the isolated virus particles.

    (A) Colony morphology of virus-carrying field strain HB58 of F. graminearum after 3 days of culture on potato dextrose agar (PDA) in the dark. (B) Viral particles observed under transmission electron microscopy. Scale bar, 50 nm. (C) One percent agarose gel electrophoresis of DNA extracted from virus particles. bp, base pair. (D) Electrophoresis analysis of enzyme-treated nucleic acid samples on 1% agarose gel. The samples were treated with DNase I, exonuclease III, S1 nuclease, and exonuclease I, respectively. (E) SDS–polyacrylamide gel electrophoresis analysis of purified virus particles from strain HB58 showing three protein bands. (F) Southern blot analyses of nucleic acids extracted from viral particles. Genomic DNA extracted from strain HB58 was used as a positive control. The forms of the viral DNA are indicated as OC dsDNA (open circular dsDNA), SC dsDNA (supercoiled dsDNA), and circular ssDNA. Three DNA fragments (probe A, probe B, and probe C, specific for DNA-A, DNA-B, and DNA-C, respectively) were PCR-amplified, labeled with DIG, and used as DNA probes. (G) Genome organization of the three components of FgGMTV1. The single ORF of each DNA component is displayed as a thick arrow. The positions of the potential stem-loop structure, common region major (CR-M) and common region stem-loop (CR-SL) are indicated. Photo credit: Pengfei Li, Institute of Plant Protection, Chinese Academy of Agricultural Sciences.

  • Fig. 2 Phylogenetic relationship of the Reps from FgGMTV1 and selected circular ssDNA viruses.

    An unrooted phylogenetic tree was constructed by a maximum-likelihood method based on multiple amino acid sequence alignments of the Rep using PhyML 3.0. Ambiguously aligned regions were removed using the online software Gap Strip/Squeeze v2.1.0. The best-fit model “RtREV + G + I + F” was selected using the Smart Model Selection technique implemented in the PhyML 3.0. Numbers at the nodes represent approximate likelihood ratio test values derived using an SH-like calculation (only values greater than 0.9 are shown). The position of FgGMTV1 is indicated by a red dot.

  • Fig. 3 Southern blot analysis of DNAs extracted from mycelia of PH-1 and nine transfectants.

    Nucleic acids (~1 μg) were loaded in each lane. The blots were probed with probe A, probe B, or probe C, respectively. The stained gel below the blot indicates equal loading of DNA. The positions of OC dsDNA, SC dsDNA, and circular ssDNA forms are indicated.

  • Fig. 4 Comparison of colony morphology, colonial diameter, growth rate, conidial production, and virulence among PH-1 and five different transfectants.

    (A) Colony morphology of PH-1 and five different transfectants after 4 days of culture on PDA in the dark. (B) Comparison of the colonial diameter among PH-1 and five different transfectants (n = 3 replicates). (C) Growth rate after 4 days on PDA (n = 3 replicates). (D) Conidia production after 5 days in O-carboxymethylcellulose liquid medium (n = 3 replicates). (E) FHB symptoms and the numbers of diseased spikelets per invaded wheat head caused by putting small equisized mycelial plugs into the glume of spikelets (n = 6 replicates). At 15 days after inoculation, there was a significant reduction in the number of diseased spikelets per invaded wheat head infected with strain A + B. The black dots indicate the inoculation positions. Different letters (a and b) in (B to E) denote significant differences (P < 0.05 determined by Tukey’s post hoc test). Photo credit: Pengfei Li, Institute of Plant Protection, Chinese Academy of Agricultural Sciences.

Supplementary Materials

  • Supplementary Materials

    A tripartite ssDNA mycovirus from a plant pathogenic fungus is infectious as cloned DNA and purified virions

    Pengfei Li, Shuangchao Wang, Lihang Zhang, Dewen Qiu, Xueping Zhou, Lihua Guo

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